Variant sulfatase enzymes with enhanced properties

Abstract

Mutated Pseudomonas aeruginosa sulfatase (PaS) enzymes with enhanced enzymatic activity as compared to the parental wild type enzyme are provided. The variant sulfatases of the invention advantageously allow for accurate analysis of bodily samples for the presence of drugs. Methods of using the mutated enzymes for hydrolysis of sulfate ester linkages are also provided, including with substrates such as opioids or steroids containing a sulfate ester linkage.

The invention claimed is: 
     
       1. A variant  Pseudomonas aeruginosa  sulfatase (PaS) enzyme comprising an amino acid substitution at an amino acid position corresponding to A139, of a parental PaS enzyme comprising the amino acid sequence shown in SEQ ID NO: 1. 
     
     
       2. The variant PaS enzyme of  claim 1 , which comprises an amino acid substitution selected from the group consisting of A139C, A139I, A139L, A139S and A139V. 
     
     
       3. The variant PaS enzyme of  claim 1 , which comprises an amino acid substitution at amino acid position A139 and at least one additional amino acid substitution at a position(s) selected from the group consisting of A75, R155, I156, K158, T160, I220, A323, L325, F328, K330 and M465. 
     
     
       4.
 The variant PaS enzyme of  claim 3 , which comprises at least two amino acid substitutions at amino acid positions selected from the group consisting of A139/F328; A139/R155; A139/I156; A139/I220; A139/L325; A139/K330; A139/M465; A139/I220/R155; and A139/I220/L325. 
 
     
     
       5. The variant PaS enzyme of  claim 3 , which comprises at least three amino acid substitutions at amino acid positions selected from the group consisting of A139/R155/L325; A139/K158/L325; A139/L325/F328; and A139/L325/M465. 
     
     
       6. The variant PaS enzyme of  claim 3 , which comprises at least four amino acid substitutions at amino acid positions selected from the group consisting of A139/R155/K158/L325; A139/R155/A323/L325; A139/R155/L325/F328; A139/R155/L325/M465; A75/A139/R155/L325; and A139/R155/T160/L325. 
     
     
       7. The variant PaS enzyme of  claim 1 , which comprises the amino acid sequence shown in SEQ ID NO: 6. 
     
     
       8. A variant  Pseudomonas aeruginosa  sulfatase (PaS) enzyme comprising an amino acid substitution as compared to a parental PaS enzyme comprising the amino acid sequence shown in SEQ ID NO: 1, wherein the amino acid substitution is selected from the group consisting of A139C, A139I, A139L, A139S and A139V. 
     
     
       9. A formulation comprising the variant PaS enzyme of  claim 1  and at least one excipient.